Project Abstract The long-term goal of this investigation is to understand cell cycle control and tumor suppression. The current investigation is focused on the function and mechanism of CUL9, a cytoplasmic E3 ubiquitin ligases that bind to p53 to maintain genomic integrity and suppress tumorigenesis. Four major findings we made over the past funding period led to this proposal. (1) Genetic analysis of Cul9 mutant mice demonstrates its functions in maintaining genomic integrity and tumor suppression. (2) Genetic analyses of Cul9-p53 double mutant mice and knock-in mutation in CUL9 disrupting p53 binding demonstrate the critical role of p53 in mediating CUL9's function. (3) CUL9 activates p53 function and promotes K63-linked p53 ubiquitylation. (4) CUL9 is a critical downstream effector of the CUL7, OBLS1 and CCDC8, three genes that are mutated in a mutually exclusive manner in 3M developmental disorder and form a protein complex. We propose three Specific Aims to test this hypothesis. Aim 1. Determine the mechanism of p53 activation by CUL9-catalyzed ubiquitylation Aim 2. Determine the mechanisms of CUL9 autoinhibition and regulation by CUL7 and CCDC8 Aim 3. Genetic analysis of Cul7-Cul9-p53 pathway This investigation includes three innovative aspects; the first E3 ligase that activates p53 and can catalyze both proteolytic and non-proteolytic ubiquitylation, a novel regulation of p53 by K63-linked polyubiquitylation, a novel regulation of CUL9 by autoinhibition and binding with a disease-linked protein complex. The strengths of this investigation include extensive use of knock-out and knock-in mutants in both cultured cells and mice, and combined multidisciplinary approaches to gain insights into both the physiological functions and biochemical mechanisms of the CUL7-CUL9-p53 pathway. Achieving these objectives will explore an emerging field? protein control by ubiquitin chain of different linkages, contribute new insights into the mechanism of the new class of RBR E3 ligases, shed new light on p53 regulation and genome integrity maintenance, and identify potential therapeutic targets for activating p53.